Fluid Based Analysis of Multiple Analytes by a Sensor Array

ABSTRACT

A system for the rapid characterization of multi-analyte fluids, in one embodiment, includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member into which a plurality of cavities may be formed. A series of chemically sensitive particles are, in one embodiment positioned within the cavities. The particles may be configured to produce a signal when a receptor coupled to the particle interacts with the analyte. Using pattern recognition techniques, the analytes within a multi-analyte fluid may be characterized.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.09/287,248 entitled “FLUID BASED ANALYSIS OF MULTIPLE ANALYTES BY ASENSOR ARRAY” filed on Apr. 7, 1999, which claims the benefit of U.S.Provisional Application No. 60/093,111 entitled “Fluid Based Analysis ofMultiple Analytes by a Sensor Array,” filed Jul. 16, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to this invention was federally supported, in part, bygrant No. 1R01GM57306-01 entitled “The Development of an ElectronicTongue” from the National Institute of Health and the U.S. Governmenthas certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and device for the detectionof analytes in a fluid. More particularly, the invention relates to thedevelopment of a sensor array system capable of discriminating mixturesof analytes, toxins, and/or bacteria in medical, food/beverage, andenvironmental solutions.

2. Brief Description of the Related Art

The development of smart sensors capable of discriminating differentanalytes, toxins, and bacteria has become increasingly important forclinical, environmental, health and safety, remote sensing, military,food/beverage and chemical processing applications. Although manysensors capable of high sensitivity and high selectivity detection havebeen fashioned for single analyte detection, only in a few selectedcases have array sensors been prepared which display solution phasemulti-analyte detection capabilities. The advantages of such arraysystems are their utility for the analysis of multiple analytes andtheir ability to be “trained” to respond to new stimuli. Such on siteadaptive analysis capabilities afforded by the array structures maketheir utilization promising for a variety of future applications. Arraybased sensors displaying the capacity to sense and identify complexvapors have been demonstrated recently using a number of distincttransduction schemes. For example, functional sensors based on SurfaceAcoustic Wave (SAW), tin oxide (SnO₂) sensors, conductive organicpolymers, and carbon black/polymer composites have been fashioned. Theuse of tin oxide sensors, for example, is described in U.S. Pat. No.5,654,497 to Hoffheins et al. These sensors display the capacity toidentify and discriminate between a variety of organic vapors by virtueof small site-to-site differences in response characteristics. Patternrecognition of the overall fingerprint response for the array serves asthe basis for an olfaction-like detection of the vapor phase analytespecies. Indeed, several commercial “electronic noses” have beendeveloped recently. Most of the well established sensing elements arebased on SnO₂ arrays which have been derivatized so as to yieldchemically distinct response properties. Arrays based on SAW crystalsyield extremely sensitive responses to vapor, however, engineeringchallenges have prevented the creation of large SAW arrays havingmultiple sensor sites. To our knowledge, the largest SAW device reportedto date possesses only 12 sensor elements. Additionally, limitedchemical diversity and the lack of understanding of the molecularfeatures of such systems makes their expansion into more complexanalysis difficult.

Other structures have been developed that are capable of identifying anddiscriminating volatile organic molecules. One structure involves aseries of conductive polymer layers deposited onto metal contactinglayers. When these sensors are exposed to volatile reagents, some of thevolatile reagents adsorb into the polymer layers, leading to smallchanges in the electrical resistance of these layers. It is the smalldifferences in the behavior of the various sites that allows for adiscrimination, identification, and quantification of the vapors. Thedetection process takes only a few seconds, and sensitivities ofpart-per-billion can be achieved with this relatively simple approach.This “electronic nose” system is described in U.S. Pat. No. 5,698,089 toLewis et al. which is incorporated by reference as if set forth herein.

Although the above described electronic nose provides an impressivecapability for monitoring volatile reagents, the system possesses anumber of undesirable characteristics that warrant the development ofalternative sensor array systems. For example, the electronic nose canbe used only for the identification of volatile reagents. For manyenvironmental, military, medical, and commercial applications, theidentification and quantification of analytes present in liquid orsolid-phase samples is necessary. Moreover, the electronic nose systemsare expensive (e.g., the Aromascan system costs about $50,000/unit) andbulky (≧1 ft³). Furthermore, the functional elements for the currentlyavailable electronic nose are composed of conductive polymer systemswhich possess little chemical selectivity for many of the analytes whichare of interest to the military and civilian communities.

One of the most commonly employed sensing techniques has exploitedcolloidal polymer microspheres for latex agglutination tests (LATs) inclinical analysis. Commercially available LATs for more than 60 analytesare used routinely for the detection of infectious diseases, illegaldrugs, and early pregnancy tests. The vast majority of these types ofsensors operate on the principle of agglutination of latex particles(polymer microspheres) which occurs when the antibody-derivatizedmicrospheres become effectively “cross-linked” by a foreign antigenresulting in the attachment to, or the inability to pass through afilter. The dye-doped microspheres are then detected colorimetricallyupon removal of the antigen carrying solution. However, the LATs lackthe ability to be utilized for multiple, real time analyte detectionschemes as the nature of the response intrinsically depends on acooperative effect of the entire collection of microspheres.

Similar to the electronic nose, array sensors that have shown greatanalytical promise are those based on the “DNA on a chip” technology.These devices possess a high density of DNA hybridization sites that areaffixed in a two-dimensional pattern on a planar substrate. To generatenucleotide sequence information, a pattern is created from unknown DNAfragments binding to various hybridization sites. Both radiochemical andoptical methods have provided excellent detection limits for analysis oflimited quantities of DNA. (Stimpson, D. I.; Hoijer, J. V.; Hsieh, W.;Jou, C.; Gardon, J.; Theriault, T.; Gamble, R.; Baldeschwieler, J. D.Proc. Natl. Acad. Sci. USA 1995, 92, 6379). Although quite promising forthe detection of DNA fragments, these arrays are generally not designedfor non-DNA molecules, and accordingly show very little sensitivity tosmaller organic molecules. Many of the target molecules of interest tocivilian and military communities, however, do not possess DNAcomponents. Thus, the need for a flexible, non-DNA based sensor is stilldesired. Moreover, while a number of prototype DNA chips containing upto a few thousand different nucleic acid probes have been described, theexisting technologies tend to be difficult to expand to a practicalsize. As a result, DNA chips may be prohibitively expensive forpractical uses.

A system of analyzing fluid samples using an array formed ofheterogeneous, semi-selective thin films which function as sensingreceptor units is described in U.S. Pat. No. 5,512,490 to Walt et al.,which is incorporated by reference as if set forth herein. Walt appearsto describe the use of covalently attached polymeric “cones” which aregrown via photopolymerization onto the distal face of fiber opticbundles. These sensor probes appear to be designed with the goal ofobtaining unique, continuous, and reproducible responses from smalllocalized regions of dye-doped polymer. The polymer appears to serve asa solid support for indicator molecules that provide information abouttest solutions through changes in optical properties. These polymersupported sensors have been used for the detection of analytes such aspH, metals, and specific biological entities. Methods for manufacturinglarge numbers of reproducible sensors, however, has yet to be developed.Moreover, no methods for acquisitions of data streams in a simultaneousmanner are commercially available with this system. Optical alignmentissues may also be problematic for these systems.

A method of rapid sample analysis for use in the diagnostic microbiologyfield is also desirable. The techniques now used for rapid microbiologydiagnostics detect either antigens or nucleic acids. Rapid antigentesting is based on the use of antibodies to recognize either the singlecell organism or the presence of infected cell material. Inherent tothis approach is the need to obtain and characterize the binding of theantibody to unique structures on the organism being tested. Since theidentification and isolation of the appropriate antibodies is timeconsuming, these techniques are limited to a single agent per testingmodule and there is no opportunity to evaluate the amount of agentpresent.

Most antibody methods are relatively insensitive and require thepresence of 10⁵ to 10⁷ organisms. The response time of antibody-antigenreactions in diagnostic tests of this type ranges from 10 to 120minutes, depending on the method of detection. The fastest methods aregenerally agglutination reactions, but these methods are less sensitivedue to difficulties in visual interpretation of the reactions.Approaches with slower reaction times include antigen recognition byantibody conjugated to either an enzyme or chromophore. These test typestend to be more sensitive, especially when spectrophotometric methodsare used to determine if an antigen-antibody reaction has occurred.These detection schemes do not, however, appear to allow thesimultaneous detection of multiple analytes on a single detectorplatform.

The alternative to antigen detection is the detection of nucleic acids.An approach for diagnostic testing with nucleic acids uses hybridizationto target unique regions of the target organism. These techniquesrequire fewer organisms (10³ to 10⁵), but require about five hours tocomplete. As with antibody-antigen reactions this approach has not beendeveloped for the simultaneous detection of multiple analytes.

The most recent improvement in the detection of microorganisms has beenthe use of nucleic acid amplification. Nucleic acid amplification testshave been developed that generate both qualitative and quantitativedata. However, the current limitations of these testing methods arerelated to delays caused by specimen preparation, amplification, anddetection. Currently, the standard assays require about five hours tocomplete. The ability to complete much faster detection for a variety ofmicroorganisms would be of tremendous importance to militaryintelligence, national safety, medical, environmental, and food areas.

It is therefore desirable that new sensors capable of discriminatingdifferent analytes, toxins, and bacteria be developed formedical/clinical diagnostic, environmental, health and safety, remotesensing, military, food/beverage, and chemical processing applications.It is further desired that the sensing system be adaptable to thesimultaneous detection of a variety of analytes to improve throughputduring various chemical and biological analytical procedures.

SUMMARY OF THE INVENTION

Herein we describe a system and method for the analysis of a fluidcontaining one or more analytes. The system may be used for eitherliquid or gaseous fluids. The system, in some embodiments, may generatepatterns that are diagnostic for both the individual analytes andmixtures of the analytes. The system in some embodiments, is made of aplurality of chemically sensitive particles, formed in an ordered array,capable of simultaneously detecting many different kinds of analytesrapidly. An aspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

In an embodiment of a system for detecting analytes, the system, in someembodiments, includes a light source, a sensor array, and a detector.The sensor array, in some embodiments, is formed of a supporting memberwhich is configured to hold a variety of chemically sensitive particles(herein referred to as “particles”) in an ordered array. The particlesare, in some embodiments, elements which will create a detectable signalin the presence of an analyte. The particles may produce optical (e.g.,absorbance or reflectance) or fluorescence/phosphorescent signals uponexposure to an analyte. Examples of particles include, but are notlimited to functionalized polymeric beads, agarous beads, dextrosebeads, polyacrylamide beads, control pore glass beads, metal oxidesparticles (e.g., silicon dioxide (SiO₂) or aluminum oxides (Al₂O₃)),polymer thin films, metal quantum particles (e.g., silver, gold,platinum, etc.), and semiconductor quantum particles (e.g., Si, Ge,GaAs, etc.). A detector (e.g., a charge-coupled device “CCD”) in oneembodiment is positioned below the sensor array to allow for the dataacquisition. In another embodiment, the detector may be positioned abovethe sensor array to allow for data acquisition from reflectance of thelight off of the particles.

Light originating from the light source may pass through the sensorarray and out through the bottom side of the sensor array. Lightmodulated by the particles may pass through the sensor array and ontothe proximally spaced detector. Evaluation of the optical changes may becompleted by visual inspection or by use of a CCD detector by itself orin combination with an optical microscope. A microprocessor may becoupled to the CCD detector or the microscope. A fluid delivery systemmay be coupled to the supporting member of the sensor array. The fluiddelivery system, in some embodiments, is configured to introduce samplesinto and out of the sensor array.

In an embodiment, the sensor array system includes an array ofparticles. The particles may include a receptor molecule coupled to apolymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles,while allowing the passage of the appropriate wavelengths of light. Thesupporting member may include a plurality of cavities. The cavities maybe formed such that at least one particle is substantially containedwithin the cavity.

In an embodiment, the optical detector may be integrated within thebottom of the supporting member, rather than using a separate detectingdevice. The optical detectors may be coupled to a microprocessor toallow evaluation of fluids without the use of separate detectingcomponents. Additionally, a fluid delivery system may also beincorporated into the supporting member. Integration of detectors and afluid delivery system into the supporting member may allow the formationof a compact and portable analyte sensing system.

A high sensitivity CCD array may be used to measure changes in opticalcharacteristics which occur upon binding of the biological/chemicalagents. The CCD arrays may be interfaced with filters, light sources,fluid delivery and micromachined particle receptacles, so as to create afunctional sensor array. Data acquisition and handling may be performedwith existing CCD technology. CCD detectors may be configured to measurewhite light, ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photo diodes,photodiode arrays, and microchannel plates may also be used.

A particle, in some embodiments, possess both the ability to bind theanalyte of interest and to create a modulated signal. The particle mayinclude receptor molecules which posses the ability to bind the analyteof interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte of interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner.

In one embodiment, a naturally occurring or synthetic receptor is boundto a polymeric bead in order to create the particle. The particle, insome embodiments, is capable of both binding the analyte(s) of interestand creating a detectable signal. In some embodiments, the particle willcreate an optical signal when bound to an analyte of interest.

A variety of natural and synthetic receptors may be used. The syntheticreceptors may come from a variety of classes including, but not limitedto, polynucleotides (e.g., aptamers), peptides (e.g., enzymes andantibodies), synthetic receptors, polymeric unnatural biopolymers (e.g.,polythioureas, polyguanidiniums), and imprinted polymers.Polynucleotides are relatively small fragments of DNA which may bederived by sequentially building the DNA sequence. Peptides includenatural peptides such as antibodies or enzymes or may be synthesizedfrom amino acids. Unnatural biopolymers are chemical structure which arebased on natural biopolymers, but which are built from unnatural linkingunits. For example, polythioureas and polyguanidiniums have a structuresimilar to peptides, but may be synthesized from diamines (i.e.,compounds which include at least two amine functional groups) ratherthan amino acids. Synthetic receptors are designed organic or inorganicstructures capable of binding various analytes.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors. Bacteria may also be detected using asimilar system. To detect, sense, and identify intact bacteria, the cellsurface of one bacteria may be differentiated from other bacteria, orgenomic material may be detected using oligonucleic receptors. Onemethod of accomplishing this differentiation is to target cell surfaceoligosaccharides (i.e., sugar residues). The use of synthetic receptorswhich are specific for oligosaccharides may be used to determine thepresence of specific bacteria by analyzing for cell surfaceoligosaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description as well as further objects, features andadvantages of the methods and apparatus of the present invention will bemore fully appreciated by reference to the following detaileddescription of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings in which:

FIG. 1 depicts a schematic of an analyte detection system;

FIG. 2 depicts a particle disposed in a cavity;

FIG. 3 depicts a sensor array;

FIG. 4A-F depicts the formation of a Fabry-Perot cavity on the back of asensor array;

FIG. 5 depicts the chemical constituents of a particle;

FIG. 6 depicts the chemical formulas of some receptor compounds;

FIG. 7 depicts a plot of the absorbance of green light vs. concentrationof calcium (Ca⁺²) for a particle which includes an o-cresolphthaleincomplexone receptor;

FIG. 8 depicts a schematic view of the transfer of energy from a firstindicator to a second indicator in the presence of an analyte;

FIG. 9 depicts a schematic of the interaction of a sugar molecule with aboronic acid based receptor.

FIG. 10 depicts various synthetic receptors;

FIG. 11 depicts a synthetic pathway for the synthesis of polythioureas;

FIG. 12 depicts a synthetic pathway for the synthesis ofpolyguanidiniums;

FIG. 13 depicts a synthetic pathway for the synthesis of diamines fromamino acids;

FIG. 14 depicts fluorescent diamino monomers;

FIG. 15 depicts a plot of counts/sec. (i.e., intensity) vs. time as thepH of a solution surrounding a particle coupled to o-cresolphthalein iscycled from acidic to basic conditions;

FIG. 16 depicts the color responses of a variety of sensing particles tosolutions of Ca⁺² and various pH levels;

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Herein we describe a system and method for the simultaneous analysis ofa fluid containing multiple analytes. The system may be used for eitherliquid or gaseous fluids. The system may generate patterns that arediagnostic for both individual analytes and mixtures of the analytes.The system, in some embodiments, is made of a combination of chemicallysensitive particles, formed in an ordered array, capable ofsimultaneously detecting many different kinds of analytes rapidly. Anaspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

System for Analysis of Analytes

Shown in FIG. 1 is an embodiment of a system for detecting analytes in afluid. The system, in some embodiments, includes a light source 110, asensor array 120 and a detector 130. The light source 110 may be a whitelight source or light emitting diodes (LED). In one embodiment, lightsource 110 may be a blue light emitting diode (LED) for use in systemsrelying on changes in fluorescence signals. For calorimetric (e.g.,absorbance) based systems, a white light source may be used. The sensorarray 120, in some embodiments, is formed of a supporting member whichis configured to hold a variety of particles 124. A detecting device 130(e.g., a charge-coupled device “CCD”) may be positioned below the sensorarray to allow for data acquisition. In another embodiment, thedetecting device 130 may be positioned above the sensor array.

Light originating from the light source 110, in some embodiments, passesthrough the sensor array 120 and out through the bottom side of thesensor array. The supporting member and the particles together, in someembodiments, provide an assembly whose optical properties are wellmatched for spectral analyses. Thus, light modulated by the particlesmay pass through the sensor array and onto the proximally spaceddetector 130. Evaluation of the optical changes may be completed byvisual inspection (e.g., with a microscope) or by use of amicroprocessor 140 coupled to the detector. For fluorescencemeasurements, a filter 135 may be placed between supporting member 120and detector 130 to remove the excitation wavelength. A fluid deliverysystem 160 may be coupled to the supporting member. The fluid deliverysystem 160 may be configured to introduce samples into and out of thesensor array.

In an embodiment, the sensor array system includes an array ofparticles. Upon the surface and within the interior region of theparticles are, in some embodiments, located a variety of receptors forinteracting with analytes. The supporting member, in some embodiments,is used to localize these particles as well as to serve as amicroenvironment in which the chemical assays can be performed. For thechemical/biological agent sensor arrays, the particles used for analysisare about 0.05-500 microns in diameter, and may actually change size(e.g., swell or shrink) when the chemical environment changes.Typically, these changes occur when the array system is exposed to thefluid stream which includes the analytes. For example, a fluid streamwhich comprises a non-polar solvent, may cause non-polar particles tochange in volume when the particles are exposed to the solvent. Toaccommodate these changes, it is preferred that the supporting memberconsist of an array of cavities which serve as micro test-tubes.

The supporting member may be made of any material capable of supportingthe particles, while allowing the passage of the appropriate wavelengthof light. The supporting member is also made of a material substantiallyimpervious to the fluid in which the analyte is present. A variety ofmaterials may be used including plastics, glass, silicon based materials(e.g., silicon, silicon dioxide, silicon nitride, etc.) and metals. Inone embodiment, the supporting member includes a plurality of cavities.The cavities may be formed such that at least one particle issubstantially contained within the cavity. Alternatively, a plurality ofparticles may be contained within a single cavity.

In an embodiment, the supporting member may consist of a strip ofplastic which is substantially transparent to the wavelength of lightnecessary for detection. A series of cavities may be formed within thestrip. The cavities may be configured to hold at least one particle. Theparticles may be contained within the strip by a transparent cover whichis configured to allow passage of the analyte containing fluid into thecavities.

In another embodiment, the supporting member may be formed using asilicon wafer as depicted in FIG. 2. The silicon wafer 210 may include asubstantially transparent layer 220 formed on the bottom surface of thewafer. The cavities 230, in one embodiment, are formed by an anisotropicetch process of the silicon wafer. In one embodiment, anisotropicetching of the silicon wafer is accomplished using a wet hydroxide etch.Photolithographic techniques may be used to define the locations of thecavities. The cavities may be formed such that the sidewalls of thecavities are substantially tapered at an angle of between about 50 to 60degrees. Formation of such angled cavities may be accomplished by wetanisotropic etching of <100> silicon. The term “<100> silicon” refers tothe crystal orientation of the silicon wafer. Other types of silicon,(e.g., <110> and <111> silicon) may lead to steeper angled sidewalls.For example, <111> silicon may lead to sidewalls formed at about 90degrees. The angled sides of the cavities in some embodiments, serve as“mirror layers” which may improve the light collection efficiency of thecavities. The etch process may be controlled so that the formed cavitiesextend through the silicon wafer to the upper surface of transparentlayer 220. While depicted as pyramidal, the cavities may be formed in anumber of shapes including but not limited to, spherical, oval, cubic,or rectangular. An advantage to using a silicon wafer for the supportmember, is that the silicon material is substantially opaque to thelight produced from the light source. Thus, the light may be inhibitedfrom passing from one cavity to adjacent cavities. In this manner, lightfrom one cavity may be inhibited from influencing the spectroscopicchanges produced in an adjacent cavity.

The silicon wafer, in some embodiments, has an area of approximately 1cm² to about 100 cm² and includes about 10¹ to about 10⁶ cavities. In anembodiment, about 100 cavities are formed in a ten by ten matrix. Thecenter to center distance between the cavities, in some embodiments, isabout 500 microns. Each of the cavities may include at least oneparticle.

The transparent layer 220 may serve as a window, allowing light of avariety of wavelengths to pass through the cavities 230 and to thedetector. Additionally, the transparent layer may serve as a platformonto which the individual particles 235 may be positioned. Thetransparent layer may be formed of silicon dioxide (SiO₂), siliconnitride (Si₃N₄) or silicon dioxide/silicon nitride multi-layer stacks.The transparent layer, in some embodiments, is deposited onto thesilicon wafer prior to the formation of the cavities.

The cavities 230 may be sized to substantially contain a particle 235.The cavities are, in some embodiments, larger than a particle. Thecavities are, in some embodiments, sized to allow facile placement andremoval of the particle within the cavities. The cavity may besubstantially larger than the particle, thus allowing the particle toswell during use. For example, a particle may have a size as depicted inFIG. 2 by particle 235. During use, contact with a fluid (e.g., asolvent) may cause the particle to swell, for example, to a sizedepicted as circle 236. In some embodiments, the cavity is sized toallow such swelling of the particle during use. A particle may bepositioned at the bottom of a cavity using, e.g., a micromanipulator.After a particle has been placed within the cavity, a transparent coverplate 240 may be placed on top of the supporting member to keep theparticle in place.

When forming an array which includes a plurality of particles, theparticles may be placed in the array in an ordered fashion using themicromanipulator. In this manner, a ordered array having a predefinedconfiguration of particles may be formed. Alternatively, the particlesmay be randomly placed within the cavities. The array may subsequentlyundergo a calibration test to determine the identity of the particle atany specified location in the supporting member.

The transparent cover plate 240, in some embodiments, is coupled to theupper surface of the silicon wafer 220 such that the particles areinhibited from becoming dislodged from the cavity. The transparent coverplate, in some embodiments, is positioned a fixed distance above thesilicon wafer, as depicted in FIG. 2, to keep the particle in place,while allowing the entrance of fluids into the cavities. The transparentcover plate, in some embodiments, is positioned at a distance above thesubstrate which is substantially less than a width of the particle. Thetransparent cover plate may be made of any material which issubstantially transparent to the wavelength of light being utilized bythe detector. The transparent cover plate may be made of plastic, glass,quartz, or silicon dioxide/silicon nitride.

In one embodiment, the transparent cover plate 240, is a thin sheet ofglass (e.g., a microscope slide cover slip). The slide may be positioneda fixed distance above the silicon wafer. Support structures 241 (SeeFIG. 2) may be placed upon the silicon wafer 210 to position thetransparent cover plate 240. The support structures may be formed from apolymer or a silicon based material. In another embodiment, a polymericsubstrate is coupled to the silicon wafer to form the support structures241 for the transparent cover plate 240. In an embodiment, a plasticmaterial with an adhesive backing (e.g., cellophane tape) is positionedon the silicon wafer 210. After the support structures 241 are placed onthe wafer the transparent cover plate 240 is placed upon the supportstructures. The support structures inhibit the transparent cover sheetfrom contacting the silicon wafer 200. In this manner, a channel isformed between the silicon wafer and the transparent cover plate whichallow the fluid to pass into the cavity, while inhibiting displacementof the particle by the fluid.

In another embodiment, the transparent cover plate 240 may be fastenedto the upper surface of the silicon wafer, as depicted in FIG. 3. Inthis embodiment, the fluid may be inhibited from entering the cavities230 by the transparent cover plate 240. To allow passage of the fluidinto the cavities, a number of channels 250 may be formed in the siliconwafer. The channels, in one embodiment, are oriented to allow passage ofthe fluid into substantially all of the cavities. When contacted withthe fluid, the particles may swell to a size which may plug thechannels. To prevent this plugging, the channels may be formed near theupper portion of the cavities, as depicted in FIG. 3. The channels, inone embodiment, are formed using standard photolithographic masking todefine the regions where the trenches are to be formed, followed by theuse of standard etching techniques. A depth of the cavity may be suchthat the particle resides substantially below the position of thechannel. In this way, the plugging of the channels due to swelling ofthe particle may be prevented.

The inner surfaces of the cavities may be coated with a material to aidthe positioning of the particles within the cavities. In one embodiment,a thin layer of gold or silver may be used to line the inner surface ofthe cavities. The gold or silver layer may act as an anchoring surfaceto anchor particles (e.g., via alkylthiol bonding). In addition, thegold or silver layer may also increase the reflectivity of the innersurface of the cavities. The increased reflectance of the surface mayenhance the analyte detection sensitivity of the system. Alternatively,polymer layers and self-assembled monolayers formed upon the innersurface of the cavities may be used to control the particle adhesioninteractions. Additional chemical anchoring methods may be used forsilicon surfaces such as those based on siloxane type reagents, whichmay be attached to Si—OH functionalities. Similarly, monomeric andpolymeric reagents attached to an interior region of the cavities can beused to alter the local wetting characteristics of the cavities. Thistype of methodology can be used to anchor the particles as well as toalter the fluid delivery characteristics of the cavity. Furthermore,amplification of the signals for the analytes may be accomplished withthis type of strategy by causing preconcentration of appropriateanalytes in the appropriate type of chemical environment.

In another embodiment, the optical detector may be integrated within thebottom transparent layer 220 of the supporting member, rather than usinga separate detecting device. The optical detectors may be formed using asemiconductor-based photodetector 255. The optical detectors may becoupled to a microprocessor to allow evaluation of fluids without theuse of separate detecting components. Additionally, the fluid deliverysystem may also be incorporated into the supporting member. Micro-pumpsand micro-valves may also be incorporated into the silicon wafer to aidpassage of the fluid through the cavities. Integration of detectors anda fluid delivery system into the supporting member may allow theformation of a compact and portable analyte sensing system. Opticalfilters may also be integrated into the bottom membrane of the cavities.These filters may prevent short wavelength excitation from producing“false” signals in the optical detection system (e.g., a CCD detectorarray) during fluorescence measurements.

A sensing cavity may be formed on the bottom surface of the supportsubstrate. An example of a sensing cavity that may be used is aFabry-Perot type cavity. Fabry-Perot cavity-based sensors may be used todetect changes in optical path length induced by either a change in therefractive index or a change in physical length of the cavity. Usingmicromachining techniques, Fabry-Perot sensors may be formed on thebottom surface of the cavity.

FIGS. 4A-F depict a sequence of processing steps for the formation of acavity and a planar top diaphragm Fabry-Perot sensor on the bottomsurface of a silicon based supporting member. A sacrificial barrierlayer 262 a/b is deposited upon both sides of a silicon supportingmember 260. The silicon supporting member 260 may be a double-sidepolished silicon wafer having a thickness ranging from about 100 μm toabout 500 μm, preferably from about 200 μm to about 400 μm, and morepreferably of about 300 μm. The barrier layer 262 a/b may be composed ofsilicon dioxide, silicon nitride, or silicon oxynitride. In oneembodiment, the barrier layer 262 a/b is composed of a stack ofdielectric materials. As depicted in FIG. 4A, the barrier layer 262 a/bis composed of a stack of dielectric materials which includes a siliconnitride layer 271 a/b and a silicon dioxide layer 272 a/b. Both layersmay be deposited using a low pressure chemical vapor deposition(“LPCVD”) process. Silicon nitride may be deposited using an LPCVDreactor by reaction of ammonia (NH₃) and dichlorosilane (SiCl₂H₂) at agas flow rate of about 3.5:1, a temperature of about 800° C., and apressure of about 220 mTorr. The silicon nitride layer 271 a/b isdeposited to a thickness in the range from about 100 Å to about 500 Å,preferably from 200 Å to about 400 Å, and more preferably of about 300Å. Silicon dioxide is may be deposited using an LPCVD reactor byreaction of silane (SiH₄) and oxygen (O₂) at a gas flow rate of about3:4, a temperature of about 450° C., and a pressure of about 110 mTorr.The silicon dioxide layer 272 a/b is deposited to a thickness in therange from about 3000 Å to about 7000 Å, preferably from 4000 Å to about6000 Å, and more preferably of about 5000 Å. The front face silicondioxide layer 272 a, in one embodiment, acts as the main barrier layer.The underlying silicon nitride layer 271 a acts as an intermediatebarrier layer to inhibit overetching of the main barrier layer duringsubsequent KOH wet anisotropic etching steps.

A bottom diaphragm layer 264 a/b is deposited upon the barrier layer 262a/b on both sides of the supporting member 260. The bottom diaphragmlayer 264 a/b may be composed of silicon nitride, silicon dioxide, orsilicon oxynitride. In one embodiment, the bottom diaphragm layer 264a/b is composed of a stack of dielectric materials. As depicted in FIG.4A, the bottom diaphragm layer 264 a/b is composed of a stack ofdielectric materials which includes a pair of silicon nitride layers 273a/b and 275 a/b surrounding a silicon dioxide layer 274 a/b. All of thelayers may be deposited using an LPCVD process. The silicon nitridelayers 273 a/b and 275 a/b have a thickness in the range from about 500Å to about 1000 Å, preferably from 700 Å to about 800 Å, and morepreferably of about 750 Å. The silicon dioxide layer 274 a/b has athickness in the range from about 3000 Å to about 7000 Å, preferablyfrom 4000 Å to about 6000 Å, and more preferably of about 4500 Å.

A cavity which will hold the particle may now be formed in thesupporting member 260. The bottom diaphragm layer 264 b and the barrierlayer 262 b formed on the back side 261 of the silicon supporting member260 are patterned and etched using standard photolithographictechniques. In one embodiment, the layers are subjected to a plasma etchprocess. The plasma etching of silicon dioxide and silicon nitride maybe performed using a mixture of carbontetrafluoride (CF₄) and oxygen(O₂). The patterned back side layers 262 b and 264 b may be used as amask for anisotropic etching of the silicon supporting member 260. Thesilicon supporting member 260, in one embodiment, is anisotropicallyetched with a 40% potassium hydroxide (“KOH”) solution at 80° C. to formthe cavity. The etch is stopped when the front side silicon nitridelayer 271 a is reached, as depicted in FIG. 4B. The silicon nitridelayer 271 a inhibits etching of the main barrier layer 272 a during thisetch process. The cavity 267 may be formed extending through thesupporting member 260. After formation of the cavity, the remainingportions of the back side barrier layer 262 b and the diaphragm layer264 b may be removed.

Etch windows 266 are formed through the bottom diaphragm layer 264 a onthe front side of the wafer. A masking layer (not shown) is formed overthe bottom diaphragm layer 264 a and patterned using standardphotolithographic techniques. Using the masking layer, etch windows 266may be formed using a plasma etch. The plasma etching of silicon dioxideand silicon nitride may be performed using a mixture ofcarbontetrafluoride (CF₄) and oxygen (O₂). The etching is continuedthrough the bottom diaphragm layer 264 a and partially into the barrierlayer 262 a. In one embodiment, the etching is stopped at approximatelyhalf the thickness of the barrier layer 262 a. Thus, when the barrierlayer 262 a is subsequently removed the etch windows 266 will extendthrough the bottom diaphragm layer 264 a, communicating with the cavity267. By stopping the etching at a midpoint of the barrier layer, voidsor discontinuities may be reduced since the bottom diaphragm is stillcontinuous due to the remaining barrier layer.

After the etch windows 266 are formed, a sacrificial spacer layer 268a/b is deposited upon the bottom diaphragm layer 264 a and within cavity267, as depicted in FIG. 4C. The spacer layer may be formed from LPCVDpolysilicon. In one embodiment, the front side deposited spacer layer268 a will also at least partially fill the etch windows 266.Polysilicon may be deposited using an LPCVD reactor using silane (SiH₄)at a temperature of about 650° C. The spacer layer 268 a/b is depositedto a thickness in the range from about 4000 Å to about 10,000 Å,preferably from 6000 Å to about 8000 Å, and more preferably of about7000 Å. The preferred thickness of the spacer layer 268 a is dependenton the desired thickness of the internal air cavity of the Fabry-Perotdetector. For example, if a Fabry-Perot detector which is to include a7000 Å air cavity between the top and bottom diaphragm layer is desired,a spacer layer having a thickness of about 7000 Å would be formed. Afterthe spacer layer has been deposited, a masking layer for etching thespacer layer 268 a (not shown) is used to define the etch regions of thespacer layer 268 a. The etching may be performed using a composition ofnitric acid (HNO₃), water, and hydrogen fluoride (HF) in a ratio of25:13:1, respectively, by volume. The lateral size of the subsequentlyformed cavity is determined by the masking pattern used to define theetch regions of the spacer layer 268 a.

After the spacer layer 268 a has been etched, the top diaphragm layer270 a/b is formed. The top diaphragm 270 a/b, in one embodiment, isdeposited upon the spacer layer 268 a/b on both sides of the supportingmember. The top diaphragm 270 a/b may be composed of silicon nitride,silicon dioxide, or silicon oxynitride. In one embodiment, the topdiaphragm 270 a/b is composed of a stack of dielectric materials. Asdepicted in FIG. 4C, the top diaphragm 270 a/b is composed of a stack ofdielectric materials which includes a pair of silicon nitride layers 283a/b and 285 a/b surrounding a silicon dioxide layer 284 a/b. All of thelayers may be deposited using an LPCVD process. The silicon nitridelayers 283 a/b and 285 a/b have a thickness in the range from about 1000Å to about 2000 Å, preferably from 1200 Å to about 1700 Å, and morepreferably of about 1500 Å. The silicon dioxide layer 284 a/b has athickness in the range from about 5000 Å to about 15,500 Å, preferablyfrom 7500 Å to about 12,000 Å, and more preferably of about 10,500 Å.

After depositing the top diaphragm 270 a/b, all of the layers stacked onthe bottom face of the supporting member (e.g., layers 268 b, 283 b, 284b, and 285 b) are removed by multiple wet and plasma etching steps, asdepicted in FIG. 4D. After these layers are removed, the now exposedportions of the barrier layer 262 a are also removed. This exposes thespacer layer 268 a which is present in the etch windows 266. The spacerlayer 268 may be removed from between the top diaphragm 270 a and thebottom diaphragm 264 a by a wet etch using a KOH solution, as depictedin FIG. 4D. Removal of the spacer material 268 a, forms a cavity 286between the top diaphragm layer 270 a and the bottom diaphragm layer 264a. After removal of the spacer material, the cavity 286 may be washedusing deionized water, followed by isopropyl alcohol to clean out anyremaining etching solution.

The cavity 286 of the Fabry-Perot sensor may be filled with a sensingsubstrate 290, as depicted in FIG. 4E. To coat the cavity 286 with asensing substrate 290, the sensing substrate may be dissolved in asolvent. A solution of the sensing substrate is applied to thesupporting member 260. The solution is believed to rapidly enter thecavity 286 through the etched windows 266 in the bottom diaphragm 264 a,aided in part by capillary action. As the solvent evaporates, a thinfilm of the sensing substrate 290 coats the inner walls of the cavity286, as well as the outer surface of the bottom diaphragm 264 a. Byrepeated treatment of the supporting member with the solution of thesensing substrate, the thickness of the sensing substrate may be varied.

In one embodiment, the sensing substrate 290 is poly(3-dodecylthiophene)whose optical properties change in response to changes in oxidationstates. The sensing substrate poly(3-dodecylthiophene) may be dissolvedin a solvent such as chloroform or xylene. In one embodiment, aconcentration of about 0.1 g of poly(3-dodecylthiophene)/mL is used.Application of the solution of poly(3-dodecylthiophene) to thesupporting member causes a thin film of poly(3-dodecylthiophene) to beformed on the inner surface of the cavity.

In some instances, the sensing substrate, when deposited within a cavityof a Fabry-Perot type detector, may cause stress in the top diaphragm ofthe detector. It is believed that when a sensing polymer coats a planartop diaphragm, extra residual stress on the top diaphragm causes thediaphragm to become deflected toward the bottom diaphragm. If thedeflection becomes to severe, sticking between the top and bottomdiaphragms may occur. In one embodiment, this stress may be relieved bythe use of supporting members 292 formed within the cavity 286, asdepicted in FIG. 4F. The supporting members 292 may be formed withoutany extra processing steps to the above described process flow. Theformation of supporting members may be accomplished by deliberatelyleaving a portion of the spacer layer within the cavity. This may beaccomplished by underetching the spacer layer (e.g., terminating theetch process before the entire etch process is finished). The remainingspacer will behave as a support member to reduce the deflection of thetop diaphragm member. The size and shape of the support members may beadjusted by altering the etch time of the spacer layer, or adjusting theshape of the etch windows 266.

In another embodiment, a high sensitivity CCD array may be used tomeasure changes in optical characteristics which occur upon binding ofthe biological/chemical agents. The CCD arrays may be interfaced withfilters, light sources, fluid delivery and micromachined particlereceptacles, so as to create a functional sensor array. Data acquisitionand handling may be performed with existing CCD technology. Data streams(e.g., red, green, blue for colorimetric assays; gray intensity forfluorescence assays) may be transferred from the CCD to a computer via adata acquisition board. Current CCDs may allow for read-out rates of 10⁵pixels per second. Thus, the entire array of particles may be evaluatedhundreds of times per second allowing for studies of the dynamics of thevarious host-guest interaction rates as well as the analyte/polymerdiffusional characteristics. Evaluation of this data may offer a methodof identifying and quantifying the chemical/biological composition ofthe test samples. CCD detectors may be configured to measure whitelight, ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photodiode, photodiodearrays, and microchannel plates may also be used. It should beunderstood that while the detector is depicted as being positioned underthe supporting member, the detector may also be positioned above thesupporting member. It should also be understood that the detectortypically includes a sensing element for detecting the spectroscopicevents and a component for displaying the detected events. The displaycomponent may be physically separated from the sensing element. Thesensing element may be positioned above or below the sensor array whilethe display component is positioned close to a user.

In one embodiment, a CCD detector may be used to record color changes ofthe chemical sensitive particles during analysis. As depicted in FIG. 1,a CCD detector 130 may be placed beneath the supporting member 120. Thelight transmitted through the cavities is captured and analyzed by theCCD detector. In one embodiment, the light is broken down into threecolor components, red, green and blue. To simplify the data, each coloris recorded using 8 bits of data. Thus, the data for each of the colorswill appear as a value between 0 and 255. The color of each chemicalsensitive element may be represented as a red, blue and green value. Forexample, a blank particle (i.e., a particle which does not include areceptor) will typically appear white. For example, when broken downinto the red, green and blue components, it is found that a typicalblank particle exhibits a red value of about 253, a green value of about250, and a blue value of about 222. This signifies that a blank particledoes not significantly absorb red, green or blue light. When a particlewith a receptor is scanned, the particle may exhibit a color change, dueto absorbance by the receptor. For example, it was found that when aparticle which includes a 5-carboxyfluorescein receptor is subjected towhite light, the particle shows a strong absorbance of blue light. TheCCD detector values for the 5-carboxyfluorescein particle exhibits a redvalue of about 254, a green value of about 218, and a blue value ofabout 57. The decrease in transmittance of blue light is believed to bedue to the absorbance of blue light by the 5-carboxyfluorescein. In thismanner, the color changes of a particle may be quantitativelycharacterized. An advantage of using a CCD detector to monitor the colorchanges is that color changes which may not be noticeable to the humaneye may now be detected.

The support array may be configured to allow a variety of detectionmodes to be practiced. In one embodiment, a light source is used togenerate light which is directed toward the particles. The particles mayabsorb a portion of the light as the light illuminates the particles.The light then reaches the detector, reduced in intensity by theabsorbance of the particles. The detector may be configure to measurethe reduction in light intensity (i.e., the absorbance) due to theparticles. In another embodiment, the detector may be placed above thesupporting member. The detector may be configured to measure the amountof light reflected off of the particles. The absorbance of light by theparticles is manifested by a reduction in the amount of light beingreflected from the cavity. The light source in either embodiment may bea white light source or a fluorescent light source.

Chemically Sensitive Particles

A particle, in some embodiments, possess both the ability to bind theanalyte of interest and to create a modulated signal. The particle mayinclude receptor molecules which posses the ability to bind the analyteof interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte of interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner. The forces which driveassociation/recognition between molecules include the hydrophobiceffect, anion-cation attraction, and hydrogen bonding. The relativestrengths of these forces depend upon factors such as the solventdielectric properties, the shape of the host molecule, and how itcomplements the guest. Upon host-guest association, attractiveinteractions occur and the molecules stick together. The most widelyused analogy for this chemical interaction is that of a “lock and key”.The fit of the key molecule (the guest) into the lock (the host) is amolecular recognition event.

A naturally occurring or synthetic receptor may be bound to a polymericresin in order to create the particle. The polymeric resin may be madefrom a variety of polymers including, but not limited to, agarous,dextrose, acrylamide, control pore glass beads, polystyrene-polyethyleneglycol resin, polystyrene-divinyl benzene resin, formylpolystyreneresin, trityl-polystyrene resin, acetyl polystyrene resin, chloroacetylpolystyrene resin, aminomethyl polystyrene-divinylbenzene resin,carboxypolystyrene resin, chloromethylated polystyrene-divinylbenzeneresin, hydroxymethyl polystyrene-divinylbenzene resin, 2-chlorotritylchloride polystyrene resin, 4-benzyloxy-2′4′-dimethoxybenzhydrol resin(Rink Acid resin), triphenyl methanol polystyrene resin,diphenylmethanol resin, benzhydrol resin, succinimidyl carbonate resin,p-nitrophenyl carbonate resin, imidazole carbonate resin, polyacrylamideresin, 4-sulfamylbenzoyl-4′-methylbenzhydrylamine-resin (Safety-catchresin), 2-amino-2-(2′-nitrophenyl) propionic acid-aminomethyl resin (ANPResin), p-benzyloxybenzyl alcohol-divinylbenzene resin (Wang resin),p-methylbenzhydrylamine-divinylbenzene resin (MBHA resin),Fmoc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine linked to resin(Knorr resin), 4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin(Rink resin), 4-hydroxymethyl-benzoyl-4′-methylbenzhydrylamine resin(HMBA-MBHA Resin), p-nitrobenzophenone oxime resin (Kaiser oxime resin),and amino-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine handlelinked to 2-chlorotrityl resin (Knorr-2-chlorotrityl resin). In oneembodiment, the material used to form the polymeric resin is compatiblewith the solvent in which the analyte is dissolved. For example,polystyrene-divinyl benzene resin will swell within non-polar solvents,but does not significantly swell within polar solvents. Thus,polystyrene-divinyl benzene resin may be used for the analysis ofanalytes within non-polar solvents. Alternatively,polystyrene-polyethylene glycol resin will swell with polar solventssuch as water. Polystyrene-polyethylene glycol resin may be useful forthe analysis of aqueous fluids.

In one embodiment, a polystyrene-polyethylene glycol-divinyl benzenematerial is used to form the polymeric resin. Thepolystyrene-polyethylene glycol-divinyl benzene resin is formed from amixture of polystyrene 375, divinyl benzene 380 andpolystyrene-polyethylene glycol 385, see FIG. 5. The polyethylene glycolportion of the polystyrene-polyethylene glycol 385, in one embodiment,may be terminated with an amine. The amine serves as a chemical handleto anchor both receptors and indicator dyes. Other chemical functionalgroups may be positioned at the terminal end of the polyethylene glycolto allow appropriate coupling of the polymeric resin to the receptormolecules or indicators.

The chemically sensitive particle, in one embodiment, is capable of bothbinding the analyte(s) of interest and creating a detectable signal. Inone embodiment, the particle will create an optical signal when bound toan analyte of interest. The use of such a polymeric bound receptorsoffers advantages both in terms of cost and configurability. Instead ofhaving to synthesize or attach a receptor directly to a supportingmember, the polymeric bound receptors may be synthesized en masse anddistributed to multiple different supporting members. This allows thecost of the sensor array, a major hurdle to the development ofmass-produced environmental probes and medical diagnostics, to bereduced. Additionally, sensor arrays which incorporate polymeric boundreceptors may be reconfigured much more quickly than array systems inwhich the receptor is attached directly to the supporting member. Forexample, if a new variant of a pathogen or a pathogen that contains agenetically engineered protein is a threat, then a new sensor arraysystem may be readily created to detect these modified analytes bysimply adding new sensor elements (e.g., polymeric bound receptors) to apreviously formed supporting member.

In one embodiment, a receptor, which is sensitive to changes in the pHof a fluid sample is bound to a polymeric resin to create a particle.That is, the receptor is sensitive to the concentration of hydrogencations (H⁺). The receptor in this case is typically sensitive to theconcentration of H⁺ in a fluid solution. The analyte of interest maytherefore be H⁺. There are many types of molecules which undergo a colorchange when the pH of the fluid is changed. For example, many types ofdyes undergo significant color changes as the pH of the fluid medium isaltered. Examples of receptors which may be used to monitor the pH of afluid sample include 5-carboxyfluorescein and alizarin complexone,depicted in FIG. 6. Each of these receptors undergoes significant colorchanges as the pH of the fluid is altered. 5-carboxyfluoresceinundergoes a change from yellow to orange as the pH of the fluid isincreased. Alizarin complexone undergoes two color changes, first fromyellow to red, then from red to blue as the pH of the fluid increases.By monitoring the change in color caused by dyes attached to a polymericparticle, the pH of a solution may be qualitatively and, with the use ofa detector (e.g., a CCD detector), quantitatively monitored.

In another embodiment, a receptor which is sensitive to presence ofmetal cations is bound to a polymeric particle to create a particle. Thereceptor in this case is typically sensitive to the concentration of oneor more metal cations present in a fluid solution. In general, coloredmolecules which will bind cations may be used to determine the presenceof a metal cation in a fluid solution. Examples of receptors which maybe used to monitor the presence of cations in a fluid sample includealizarin complexone and o-cresolphthalein complexone, see FIG. 6. Eachof these receptors undergoes significant color changes as theconcentration of a specific metal ion in the fluid is altered. Alizarincomplexone is particularly sensitive to lanthanum ions. In the absenceof lanthanum, alizarin complexone will exhibit a yellow color. As theconcentration of lanthanum is increased, alizarin complexone will changeto a red color. o-Cresolphthalein complexone is particularly sensitiveto calcium ions. In the absence of calcium, o-cresolphthalein complexoneis colorless. As the concentration of calcium is increased,o-cresolphthalein complexone will change to a blue color. By monitoringthe change in color of metal cation sensitive receptors attached to apolymeric particle, the presence of a specific metal ion may bequalitatively and, with the use of a detector (e.g., a CCD detector),quantitatively monitored.

Referring to FIG. 7, a graph of the absorbance of green light vs.concentration of calcium (Ca⁺²) is depicted for a particle whichincludes an o-cresolphthalein complexone receptor. As the concentrationof calcium is increased, the absorbance of green light increases in alinear manner up to a concentration of about 0.0006 M. A concentrationof 0.0006 M is the solubility limit of calcium in the fluid, thus nosignificant change in absorbance is noted after this point. The linearrelationship between concentration and absorbance allows theconcentration of calcium to be determined by measuring the absorbance ofthe fluid sample.

In one embodiment, a detectable signal may be caused by the altering ofthe physical properties of an indicator ligand bound to the receptor orthe polymeric resin. In one embodiment, two different indicators areattached to a receptor or the polymeric resin. When an analyte iscaptured by the receptor, the physical distance between the twoindicators may be altered such that a change in the spectroscopicproperties of the indicators is produced. A variety of fluorescent andphosphorescent indicators may be used for this sensing scheme. Thisprocess, known as Forster energy transfer, is extremely sensitive tosmall changes in the distance between the indicator molecules.

For example, a first fluorescent indicator 320 (e.g., a fluoresceinderivative) and a second fluorescent indictor 330 (e.g., a rhodaminederivative) may be attached to a receptor 300, as depicted in FIG. 8.When no analyte is present short wavelength excitation 310 may excitethe first fluorescent indicator 320, which fluoresces as indicated by312. The short wavelength excitation, however, may cause little or nofluorescence of the second fluorescent indicator 330. After binding ofanalyte 350 to the receptor, a structural change in the receptormolecule may bring the first and second fluorescent indicators closer toeach other. This change in intermolecular distance may allow the excitedfirst indicator 320 to transfer a portion of its fluorescent energy 325to the second fluorescent indicator 330. This transfer in energy may bemeasured by either a drop in energy of the fluorescence of the firstindicator molecule 320, or the detection of increased fluorescence 314by the second indicator molecule 330.

Alternatively, the first and second fluorescent indicators may initiallybe positioned such that short wavelength excitation, may causefluorescence of both the first and second fluorescent indicators, asdescribed above. After binding of analyte 350 to the receptor, astructural change in the receptor molecule may cause the first andsecond fluorescent indicators to move further apart. This change inintermolecular distance may inhibit the transfer of fluorescent energyfrom the first indicator 320 to the second fluorescent indicator 330.This change in the transfer of energy may be measured by either a dropin energy of the fluorescence of the second indicator molecule 330, orthe detection of increased fluorescence by the first indicator molecule320.

In another embodiment, an indicator ligand may be preloaded onto thereceptor. An analyte may then displace the indicator ligand to produce achange in the spectroscopic properties of the particles. In this case,the initial background absorbance is relatively large and decreases whenthe analyte is present. The indicator ligand, in one embodiment, has avariety of spectroscopic properties which may be measured. Thesespectroscopic properties include, but are not limited to, ultravioletabsorption, visible absorption, infrared absorption, fluorescence, andmagnetic resonance. In one embodiment, the indicator is a dye havingeither a strong fluorescence, a strong ultraviolet absorption, a strongvisible absorption, or a combination of these physical properties.Examples of indicators include, but are not limited to,carboxyfluorescein, ethidium bromide, 7-dimethylamino-4-methylcoumarin,7-diethylamino-4-methylcoumarin, eosin, erythrosin, fluorescein, OregonGreen 488, pyrene, Rhodamine Red, tetramethylrhodamine, Texas Red,Methyl Violet, Crystal Violet, Ethyl Violet, Malachite green, MethylGreen, Alizarin Red S, Methyl Red, Neutral Red,o-cresolsulfonephthalein, o-cresolphthalein, phenolphthalein, AcridineOrange, B-naphthol, coumarin, and a-naphthionic acid. When the indicatoris mixed with the receptor, the receptor and indicator interact witheach other such that the above mentioned spectroscopic properties of theindicator, as well as other spectroscopic properties may be altered. Thenature of this interaction may be a binding interaction, wherein theindicator and receptor are attracted to each other with a sufficientforce to allow the newly formed receptor-indicator complex to functionas a single unit. The binding of the indicator and receptor to eachother may take the form of a covalent bond, an ionic bond, a hydrogenbond, a van der Waals interaction, or a combination of these bonds.

The indicator may be chosen such that the binding strength of theindicator to the receptor is less than the binding strength of theanalyte to the receptor. Thus, in the presence of an analyte, thebinding of the indicator with the receptor may be disrupted, releasingthe indicator from the receptor. When released, the physical propertiesof the indicator may be altered from those it exhibited when bound tothe receptor. The indicator may revert back to its original structure,thus regaining its original physical properties. For example, if afluorescent indicator is attached to a particle that includes areceptor, the fluorescence of the particle may be strong beforetreatment with an analyte containing fluid. When the analyte interactswith the particle, the fluorescent indicator may be released. Release ofthe indicator may cause a decrease in the fluorescence of the particle,since the particle now has less indicator molecules associated with it.

An example of this type of system is illustrated by the use of a boronicacid substituted resin 505 as a particle. Prior to testing, the boronicacid substituted resin 505 is treated with a sugar 510 which is taggedwith an indicator (e.g., resorufin) as depicted in FIG. 9. The sugar 510binds to the boronic acid receptor 500 imparting a color change to theboronic substituted resin 505 (yellow for the resorufin tagged sugar).When the boronic acid resin 505 is treated with a fluid sample whichincludes a sugar 520, the tagged sugar 510 may be displaced, causing adecrease in the amount of color produced by the boronic acid substitutedresin 505. This decrease may be qualitatively or, with the use of adetector (e.g., a CCD detector), quantitatively monitored.

In another embodiment, a designed synthetic receptor may be used. In oneembodiment, a polycarboxylic acid receptor may be attached to apolymeric resin. The polycarboxylic receptors are discussed in U.S.patent application Ser. No. 08/950,712 which is incorporated herein byreference.

In an embodiment, the analyte molecules in the fluid may be pretreatedwith an indicator ligand. Pretreatment may involve covalent attachmentof an indicator ligand to the analyte molecule. After the indicator hasbeen attached to the analyte, the fluid may be passed over the sensingparticles. Interaction of the receptors on the sensing particles withthe analytes may remove the analytes from the solution. Since theanalytes include an indicator, the spectroscopic properties of theindicator may be passed onto the particle. By analyzing the physicalproperties of the sensing particles after passage of an analyte stream,the presence and concentration of an analyte may be determined.

For example, the analytes within a fluid may be derivatized with afluorescent tag before introducing the stream to the particles. Asanalyte molecules are adsorbed by the particles, the fluorescence of theparticles may increase. The presence of a fluorescent signal may be usedto determine the presence of a specific analyte. Additionally, thestrength of the fluorescence may be used to determine the amount ofanalyte within the stream.

Receptors

A variety of natural and synthetic receptors may be used. The syntheticreceptors may come from a variety of classes including, but not limitedto, polynucleotides (e.g., aptamers), peptides (e.g., enzymes andantibodies), synthetic receptors, polymeric unnatural biopolymers (e.g.,polythioureas, polyguanidiniums), and imprinted polymers, some of whichare generally depicted in FIG. 10. Natural based synthetic receptorsinclude receptors which are structurally similar to naturally occurringmolecules. Polynucleotides are relatively small fragments of DNA whichmay be derived by sequentially building the DNA sequence. Peptides maybe synthesized from amino acids. Unnatural biopolymers are chemicalstructure which are based on natural biopolymers, but which are builtfrom unnatural linking units. Unnatural biopolymers such aspolythioureas and polyguanidiniums may be synthesized from diamines(i.e., compounds which include at least two amine functional groups).These molecules are structurally similar to naturally occurringreceptors, (e.g., peptides). Some diamines may, in turn, be synthesizedfrom amino acids. The use of amino acids as the building blocks forthese compounds allow a wide variety of molecular recognition units tobe devised. For example, the twenty natural amino acids have side chainsthat possess hydrophobic residues, cationic and anionic residues, aswell as hydrogen bonding groups. These side chains may provide a goodchemical match to bind a large number of targets, from small moleculesto large oligosaccharides. Amino acid based peptides, polythioureas, andpolyguanidiniums are depicted in FIG. 10.

Techniques for the building of DNA fragments and polypeptide fragmentson a polymer particle are well known. Techniques for the immobilizationof naturally occurring antibodies and enzymes on a polymeric resin arealso well known. The synthesis of polythioureas upon a resin particlemay be accomplished by the synthetic pathway depicted in FIG. 11. Theprocedure may begin by deprotection of the terminal tBoc protectinggroup on an amino acid coupled to a polymeric particle. Removal of theprotecting group is followed by coupling of the rigid spacer 410 to theresulting amine 405 using diisopropylcarbodiimide (DIC) and1-hydroxybenzotriazole hydrate (HOBT). The spacer group may inhibitformation of a thiazolone by reaction of the first amino acids withsubsequently formed thioureas. After the spacer group is coupled to theamino acid, another tBoc deprotection is performed to remove the spacerprotecting group, giving the amine 415. At this point, monomer may beadded incrementally to the growing chain, each time followed by a tBocdeprotection. The addition of a derivative of the diamine 420 (e.g., anisothiocyanate) to amine 415 gives the mono-thiourea 425. The additionof a second thiourea substituent is also depicted. After the addition ofthe desired number of monomers, a solution of benzylisothiocyanate oracetic anhydride may be added to cap any remaining amines on the growingoligomers. Between 1 to 20 thioureas groups may be formed to produce asynthetic polythiourea receptor.

The synthesis of polyguanidiniums may be accomplished as depicted inFIG. 12. In order to incorporate these guanidinium groups into thereceptor, the coupling of a thiourea with a terminal amine in thepresence of Mukaiyama's reagent may be utilized. The coupling of thefirst thiourea diamine 430 with an amino group of a polymeric particlegives the mono-guanidinium 434. Coupling of the resultingmono-guanidinium with a second thiourea diamine 436 gives adi-guanidinium 438. Further coupling may create a tri-guanidinium 440.Between 1 to 20 guanidinium groups may be formed to produce a syntheticpolyguanidinium receptor.

The above described methods for making polythioureas andpolyguanidiniums are based on the incorporation of diamines (i.e.,molecules which include at least two amine functional groups) into theoligomeric receptor. The method may be general for any compound havingat least two amino groups. In one embodiment, the diamine may be derivedfrom amino acids. A method for forming diamines from amino acids isshown in FIG. 13. Treatment of a protected amino acid 450 withborane-THF reduces the carboxylic acid portion of the amino acid to theprimary alcohol 452. The primary alcohol is treated with phthalimideunder Mitsunobu conditions (PPh₃/DEAD). The resulting compound 454 istreated with aqueous methylamine to form the desired monoprotecteddiamine 456. The process may be accomplished such that the enantiomericpurity of the starting amino acid is maintained. Any natural orsynthetic amino acid may be used in the above described method.

The three coupling strategies used to form the respective functionalgroups may be completely compatible with each other. The capability tomix linking groups (amides, thioureas, and guanidiniums) as well as theside chains (hydrophobic, cationic, anionic, and hydrogen bonding) mayallow the creation of a diversity in the oligomers that is beyond thediversity of receptors typically found with natural biologicalreceptors. Thus, we may produce ultra-sensitive and ultra-selectivereceptors which exhibit interactions for specific toxins, bacteria, andenvironmental chemicals. Additionally, these synthetic schemes may beused to build combinatorial libraries of particles for use in the sensorarray.

In an embodiment, the indicator ligand may be incorporated intosynthetic receptors during the synthesis of the receptors. The ligandmay be incorporated into a monomeric unit, such as a diamine, that isused during the synthesis of the receptor. In this manner, the indicatormay be covalently attached to the receptor in a controlled position. Byplacing the indicator within the receptor during the synthesis of thereceptor, the positioning of the indicator ligand within the receptormay be controlled. This control may be difficult to achieve aftersynthesis of the receptor is completed.

In one embodiment, a fluorescent group may be incorporated into adiamine monomer for use in the synthetic sequences. Examples ofmonomeric units which may be used for the synthesis of a receptor aredepicted in FIG. 14. The depicted monomers include fluorescent indicatorgroups. After synthesis, the interaction of the receptor with theanalyte may induce changes in the spectroscopic properties of themolecule. Typically, hydrogen bonding or ionic substituents on thefluorescent monomer involved in analyte binding have the capacity tochange the electron density and/or rigidity of the fluorescent ringsystem, thereby causing observable changes in the spectroscopicproperties of the indicator. For fluorescent indicators such changes maybe exhibited as changes in the fluorescence quantum yield, maximumexcitation wavelength, and/or maximum emission wavelength. This approachdoes not require the dissociation of a preloaded fluorescent ligand,which may be limited in response time by k_((off))). While fluorescentligands are shown here, it is to be understood that a variety of otherligand may be used including calorimetric ligands.

In another embodiment, two fluorescent monomers for signaling may beused for the synthesis of the receptor. For example, compound 470 (aderivative of fluorescein) and compound 475 (a derivative of rhodamine),depicted in FIG. 14, may both be incorporated into a synthetic receptor.Compound 470 contains a common colorimetric/fluorescent probe that will,in some embodiments, send out a modulated signal upon analyte binding.The modulation may be due to resonance energy transfer to compound 475.When an analyte binds to the receptor, structural changes in thereceptor may alter the distance between monomeric units 470 and 475. Itis well known that excitation of fluorescein can result in emission fromrhodamine when these molecules are oriented correctly. The efficiency ofresonance energy transfer from monomers 470 to 475 will depend stronglyupon the presence of analyte binding; thus, measurement of rhodaminefluorescence intensity (at a substantially longer wavelength thanfluorescein fluorescence) may serve as an indicator of analyte binding.To greatly improve the likelihood of a modulatory fluorescein-rhodamineinteraction, multiple rhodamine tags may be attached at different sitesalong a receptor molecule without substantially increasing backgroundrhodamine fluorescence (only rhodamine very close to fluorescein willyield appreciable signal). This methodology may be applied to a numberof alternate fluorescent pairs.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors including both small and medium sizemolecules. For example, it is known that nerve gases typically producephosphate structures upon hydrolysis in water. The presence of moleculeswhich contain phosphate functional groups may be detected usingpolyguanidiniums. Nerve gases which have contaminated water sources maybe detected by the use of the polyguanidinium receptors described above.

In order to identify, sense, and quantitate the presence of variousbacteria using the proposed micro-machined sensor, two strategies may beused. First, small molecule recognition and detection may be exploited.Since each bacteria possesses a unique and distinctive concentration ofthe various cellular molecules, such as DNA, proteins, metabolites, andsugars, the fingerprint (i.e., the concentration and types of DNA,proteins, metabolites, and sugars) of each organism is expected to beunique. Hence, the analytes obtained from whole bacteria or broken downbacteria may be used to determine the presence of specific bacteria. Aseries of receptors specific for DNA molecules, proteins, metabolites,and sugars may be incorporated into an array. A solution containingbacteria, or more preferably broken down bacteria, may be passed overthe array of particles. The individual cellular components of thebacteria may interact in a different manner with each of the particles.This interaction will provide a pattern within the array which may beunique for the individual bacteria. In this manner, the presence ofbacteria within a fluid may be determined.

In another embodiment, bacteria may be detected as whole entities, asfound in ground water, aerosols, or blood. To detect, sense, andidentify intact bacteria, the cell surface of one bacteria may bedifferentiated from other bacteria. One method of accomplishing thisdifferentiation is to target cell surface oligosaccharides (i.e. sugarresidues). Each bacterial class (gram negative, gram positive, etc.)displays a different oligosaccharide on their cell surfaces. Theoligosaccharide, which is the code that is read by other cells giving anidentification of the cell, is part of the cell-cell recognition andcommunication process. The use of synthetic receptors which are specificfor oligosaccharides may be used to determine the presence of specificbacteria by analyzing for the cell surface oligosaccharides.

In another embodiment, the sensor array may be used to optimize whichreceptor molecules should be used for a specific analyte. An array ofreceptors may be placed within the cavities of the supporting member anda stream containing an analyte may be passed over the array. Thereaction of each portion of the sensing array to the known analyte maybe analyzed and the optimal receptor determined by determining whichparticle, and therefore which receptor, exhibits the strongest reactiontoward the analyte. In this manner, a large number of potentialreceptors may be rapidly scanned. The optimal receptor may then beincorporated into a system used for the detection of the specificanalyte in a mixture of analytes.

It should be emphasized that although some particles may be purposefullydesigned to bind to important species (biological agents, toxins, nervegasses, etc.), most structures will possess nonspecific receptor groups.One of the advantages associated with the proposed sensor array is thecapacity to standardize each array of particles via exposure to variousanalytes, followed by storage of the patterns which arise frominteraction of the analytes with the particles. Therefore, there may notbe a need to know the identity of the actual receptor on each particle.Only the characteristic pattern for each array of particles isimportant. In fact, for many applications it may be less time consumingto place the various particles into their respective holders withouttaking precautions to characterize the location associated with thespecific particles. When used in this manner, each individual sensorarray may require standardization for the type of analyte to be studied.

On-site calibration for new or unknown toxins may also be possible withthis type of array. Upon complexation of an analyte, the localmicroenvironment of each indicator may change, resulting in a modulationof the light absorption and/or emission properties. The use of standardpattern recognition algorithms completed on a computer platform mayserves as the intelligence factor for the analysis. The “fingerprint”like response evoked from the simultaneous interactions occurring atmultiple sites within the substrate may be used to identify the speciespresent in unknown samples.

The above described sensor array system offers a number of distinctadvantages over exiting technologies. One advantage is that “real time”detection of analytes may be performed. Another advantage is that thesimultaneous detection of multiple analytes may be realized. Yet anotheradvantage is that the sensor array system allows the use of syntheticreagents as well as biologically produced reagents. Synthetic reagentstypically have superior sensitivity and specificity toward analytes whencompared to the biological reagents. Yet another advantage is that thesensor array system may be readily modified by simply changing theparticles which are placed within the sensor array. Thisinterchangability may also reduce production costs.

EXAMPLES 1. The Determination of pH Using a Chemically SensitiveParticle

Shown in FIG. 15 is the magnitude of the optical signal transmittedthrough a single polymer particle derivatized with o-cresolphthalein.Here, a filter is used to focus the analysis on those wavelengths whichthe dye absorbs most strongly (i.e., about 550 nm). Data is provided forthe particle as the pH is cycled between acid and basic environments. Inacidic media (i.e., at times of 100-150 seconds and 180-210 seconds),the particle is clear and the system yields large signals (up to greaterthan 300,000 counts) at the optical detector. Between times of 0-100 and150-180 seconds, the solution was made basic. Upon raising the pH (i.e.,making the solution more basic), the particle turns purple in color andthe transmitted green light is greatly diminished. Large signalreductions are recorded under such circumstances. The evolution of thesignal changes show that the response time is quite rapid, on the orderof 10 seconds. Furthermore, the behavior is highly reproducible.

2. The Simultaneous Detection of Ca⁺², Ce⁺³, and pH by a Sensor ArraySystem

The synthesis of four different particles was accomplished by coupling avariety of indictor ligands to a polyethylene glycol-polystyrene(“PEG-PS”) resin particle. The PEG-PS resin particles were obtained fromNovabiochem Corp., La Jolla, Calif. The particles have an averagediameter of about 130 μm when dry and about 250 μm when wet. Theindicator ligands of fluorescein, o-cresolphthalein complexone, andalizarin complexone were each attached to PEG-PS resin particles using adicyclohexylcarbodiimide (DCC) coupling between a terminal resin boundamine and a carboxylic acid on the indicator ligand.

These synthetic receptors, localized on the PEG-PS resin to createsensing particles, were positioned within micromachined wells formed insilicon/silicon nitride wafers, thus confining the particles toindividually addressable positions on a multicomponent chip. These wellswere sized to hold the particles in both swollen and unswollen states.Rapid introduction of the test fluids can be accomplished using thesestructures while allowing spectrophotometric assays to probe for thepresence of analytes. For the identification and quantification ofanalyte species, changes in the light absorption and light emissionproperties of the immobilized resin particles can be exploited, althoughonly identification based upon absorption properties are discussed here.Upon exposure to analytes, color changes for the particles were found tobe 90% complete within one minute of exposure, although typically onlyseconds were required. To make the analysis of the colorimetric changesefficient, rapid, and sensitive, a charge-coupled-device (CCD) wasdirectly interfaced with the sensor array. Thus, data streams composedof red, green, and blue (RGB) light intensities were acquired andprocessed for each of the individual particle elements. The red, blue,and green responses of the particles to various solutions aregraphically depicted in FIG. 16.

The true power of the described bead sensor array occurs whensimultaneous evaluation of multiple chemically distinct bead structuresis completed. A demonstration of the capacity of five different beads isprovided in FIG. 16. In this case, blank, alizarin, o-cresol phthalein,fluorescein, and alizarin-Ce3+ complex derivatized beads serve as amatrix for subtle differentiation of chemical environments. The blankbead is simply a polystyrene sphere with no chemical derivatization. Thebead derivatized with o-cresolphthalein responds to Ca+2 at pHs valuesaround 10.0. The binding of calcium is noted from the large green colorattenuation noted for this dye while exposed to the cation. Similarly,the fluorescein derivatized bead acts as a pH sensor. At pHs below 7.4it is light yellow, but at higher pHs it turns dark orange. Interesting,the alizarin complexone plays three distinct roles. First, it acts as aproton sensor yielding a yellow color at pHs below 4.5, orange is notedat pHs between 4.5 and 11.5, and at pHs above 11.5 a blue hue isobserved. Second, it functions as a sensor for lanthanum ions at lowerpHs by turning yellow to orange. Third, the combination of both fluorideand lanthanum ions results in yellow/orange coloration.

The analysis of solutions containing various amount of Ca⁺² or F⁻ atvarious pH levels was performed using alizarin complexone,o-cresolphthalein complexone, 5-carboxy fluorescein, and alizarin-Ce³⁺complex. A blank particle in which the terminal amines of a PEG-PS resinparticle have been acylated was also used. In this example, the presenceof Ca⁺² (0.1 M Ca(NO₃)₂) was analyzed under conditions of varying pH.The pH was varied to values of 2, 7, and 12, all buffered by a mixtureof 0.04 M phosphate, 0.04 M acetate, and 0.04 M borate. The RGB patternsfor each sensor element in all environments were measured. The beadderivatized with o-cresolphthalein responds to Ca⁺² at pH values around12. Similarly, the 5-carboxy fluorescein derivatized bead acts as a pHsensor. At pHs below 7.4 it is light yellow, but at higher pHs it turnsdark orange. Interesting, the alizarin complexone plays three distinctroles. First, it acts as a proton sensor yielding a yellow color at pHsbelow 4.5, orange is noted at pHs between 4.5 and 11.5, and at pHs above11.5 a blue hue is observed. Second, it functions as a sensor forlanthanum ions at lower pHs by turning yellow to orange. Third, thecombination of both fluoride and lanthanum ions results in yellow/orangecoloration.

This example demonstrates a number of important factors related to thedesign, testing, and functionality of micromachined array sensors forsolution analyses. First, derivatization of polymer particles with bothcolorimetric and fluorescent dyes was completed. These structures wereshown to respond to pH and Ca²⁺. Second, response times well under 1minute were found. Third, micromachined arrays suitable both forconfinement of particles, as well as optical characterization of theparticles, have been prepared. Fourth, integration of the test bedarrays with commercially available CCD detectors has been accomplished.Finally, simultaneous detection of several analytes in a mixture wasmade possible by analysis of the RGB color patterns created by thesensor array.

3. The Detection of Sugar Molecules Using a Boronic Acid Based Receptor

A series of receptors were prepared with functionalities that associatestrongly with sugar molecules, as depicted in FIG. 9. In this case, aboronic acid sugar receptor 500 was utilized to demonstrate thefunctionality of a new type of sensing scheme in which competitivedisplacement of a resorufin derivatized galactose sugar molecule wasused to assess the presence (or lack thereof) of other sugar molecules.The boronic acid receptor 500 was formed via a substitution reaction ofa benzylic bromide. The boronic acid receptor was attached to apolyethylene glycol-polystyrene (“PEG-PS”) resin particle at the “R”position. Initially, the boronic acid derivatized particle was loadedwith resorufin derivatized galactose 510. Upon exposure of the particleto a solution containing glucose 520, the resorufin derivatizedgalactose molecules 510 are displaced from the particle receptor sites.Visual inspection of the optical photographs taken before and afterexposure to the sugar solution show that the boron substituted resin iscapable of sequestering sugar molecules from an aqueous solution.Moreover, the subsequent exposure of the colored particles to a solutionof a non-tagged sugar (e.g., glucose) leads to a displacement of thebound colored sugar reporter molecule. Displacement of this moleculeleads to a change in the color of the particle. The sugar sensor turnsfrom dark orange to yellow in solutions containing glucose. Theparticles were also tested in conditions of varying pH. It was notedthat the color of the particles changes from dark orange to yellow asthe pH is varied from low pH to high pH.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1-135. (canceled)
 136. A method of sensing an analyte in a fluidcomprising: passing a fluid over a sensor array, the sensor arraycomprising at least one particle positioned within a cavity of asupporting member; monitoring a spectroscopic change of the particle asthe fluid is passed over the sensor array, wherein the spectroscopicchange is caused by the interaction of the analyte with the particle.137. The method of claim 136, wherein the spectroscopic change comprisesa change in absorbance of the particle.
 138. The method of claim 136,wherein the spectroscopic change comprises a change in fluorescence ofthe particle.
 139. The method of claim 136, wherein the spectroscopicchange comprises a change in phosphorescence of the particle.
 140. Themethod of claim 136, wherein the analyte is a proton atom, and whereinthe spectroscopic change is produced when the pH of the fluid is varied,and wherein monitoring the spectroscopic change of the particle allowsthe pH of the fluid to be determined.
 141. The method of claim 136,wherein the analyte is a metal cation, and wherein the spectroscopicchange is produced in response to the presence of the metal cation inthe fluid.
 142. The method of claim 136, wherein the analyte is ananion, and wherein the spectroscopic change is produced in response tothe presence of the anion in the fluid.
 143. The method of claim 136,wherein the analyte is a DNA molecule, and wherein the spectroscopicchange is produced in response to the presence of the DNA molecule inthe fluid.
 144. The method of claim 136, wherein the analyte is aprotein, and wherein the spectroscopic change is produced in response tothe presence of the protein in the fluid.
 145. The method of claim 136,wherein the analyte is a metabolite, and wherein the spectroscopicchange is produced in response to the presence of the metabolite in thefluid.
 146. The method of claim 136, wherein the analyte is a sugar, andwherein the spectroscopic change is produced in response to the presenceof the sugar in the fluid.
 147. The method of claim 136, wherein theanalyte is a bacteria, and wherein the spectroscopic change is producedin response to the presence of the bacteria in the fluid.
 148. Themethod of claim 136, wherein the particle comprises a receptor coupledto a polymeric resin, and further comprising exposing the particle to anindicator prior to passing the fluid over the sensor array.
 149. Themethod of claim 148, wherein a binding strength of the indicator to thereceptor is less than a binding strength of the analyte to the receptor.150. The method of claim 148, wherein the indicator is a fluorescentindicator.
 151. The method of claim 136, further comprising treating thefluid with an indicator prior to passing the fluid over the sensorarray, wherein the indicator is configured to couple with the analyte.152. The method of claim 136, wherein the analyte is bacteria andfurther comprising breaking down the bacteria prior to passing the fluidover the sensor array.
 153. The method of claim 136, wherein monitoringthe spectroscopic change is performed with a CCD device.
 154. The methodof claim 136, further comprising measuring the intensity of thespectroscopic change, and further comprising calculating theconcentration of the analyte based on the intensity of the spectroscopicchange. 155.-172. (canceled)